Angiogenesis is a biological process of generating new blood vessels into a tissue or an organ. Under normal physiological conditions, humans or animals undergo angiogenesis only in very specific restricted situations. For example, angiogenesis is normally observed in wound healing, fetal and embryonic development and formation of the corpus luteum, endometrium and placenta. It is widely accepted that new vessel growth is tightly controlled by many angiogenic regulators and the switch of the angiogenesis phenotype depends on the net balance between up-regulation of angiogenic stimulators and down-regulation of angiogenic suppressors. The control of angiogenesis has been found to be altered in certain disease states and, in many cases, the pathological damage associated with the disease is related to the uncontrolled angiogenesis.
Both controlled and uncontrolled angiogenesis are thought to proceed in a similar manner. Endothelial cells and pericytes surrounded by a basement membrane, form capillary blood vessels. Angiogenesis begins with the erosion of the basement membrane by enzymes released by endothelial cells and leukocytes. The endothelial cells, which line the lumen of blood vessels, then protrude through the basement membrane. Angiogenic stimulants induce the endothelial cells to migrate through the eroded basement membrane. The migrating cells form a “sprout” off the parent blood vessel, where the endothelial cells undergo mitosis and proliferate. The endothelial sprouts merge with each other to form capillary loops, creating the new blood vessel.
In disease states where an imbalance of the angiogenic process is encountered, increasing or inhibiting angiogenesis could avert the corresponding body damages.
In many other situations, particularly when preventing or treating cancers is sought, a down-regulation of angiogenesis is desired.
Various substances are already known that prevent deregulation of angiogenesis, most of them inhibiting angiogenesis.
Until now, at least ten endogenous angiogenic inhibitors have been identified in the art. One such molecule is angiostatin, which consists of the plasminogen kringle domains I through IV. Also, apolipoprotein (a), one of the proteins having kringle structures, is a candidate for a novel angiogenesis inhibitor.
Several other kinds of compounds have been used to prevent angiogenesis. For example, Taylor et al. have used protamine to inhibit angiogenesis, although its toxicity limits its practical use as a therapeutic agent (Taylor et al., 1982, Nature, Vol. 297 : 307). Folkman et al. have disclosed the use of heparin and steroids to control angiogenesis (Folkman et al., 1983, Science, Vol. 221 : 719). Other agents which have been used to inhibit angiogenesis include ascorbic acid ethers and related compounds (Japanese Patent Kokai Tokkyo Koho No 58-131978). Also, a fungal product, fumagillin, is a potent angiostatic agent in vitro, as well as its synthetic derivative O-substituted fumagillin.
The United States Food and Drug Administration has granted the first marketing authorization for an anti-angiogenic therapeutic agent, which is termed bevacizumab. Bevacizumab is a humanized antibody directed against the angiogenic factor VEGF. Bevacizumab prevents the binding of VEGF to its effector receptor and has been initially used for treating colorectal cancer.
When taking into account the wide diversity of conditions or diseases that are caused by a deregulation of angiogenesis, or where deregulation of angiogenesis is involved, as well as the severity of several of these diseases, it flows that here is a high need in the art for the provision of novel therapeutically active substances that would allow circumventing physiological situations associated with a deregulation of angiogenesis, typically pathologically strong angiogenesis activity, and especially in cancer.
There is also a need in the art for new methods that would enable the screening of candidate substances for their angiogenesis regulation potency.
The N-methyl-D-Aspartate Receptor (NMDAR) is a ligand-gated and voltage-dependent channel belonging to the ionotropic glutamate receptor family5. It forms an heterotetrameric ion channel across the cell membrane and is composed of two GluN1 subunits (encoded by the GRIN1 gene), obligatory subunits to form functional NMDAR, containing a glycine or D-serine binding site, mainly associated to two GluN2 subunits either GluN2A, GluN2B, GluN2C or GluN2D subunits comprising the glutamate binding site, and modulating channel properties5. It is activated by both glutamate and glycine or D-serine, and in a context of membrane depolarization necessary to release magnesium ion acting as a channel blocker at resting potential. Conventional NMDAR has a major role in the central nervous system (CNS) as a key player of the excitatory synaptic neuronal communication5. Indeed, NMDAR distributes at the cell membrane in cell-cell contact sites either synaptic (defined by a close contact between neurons processes) or extrasynaptic sites (associated to close contacts mainly between neurons and microglial cells or astrocytes)6. Dysregulation of the NMDAR-mediated glutamatergic communication has been pointed out in many disorders in the CNS i.e. neurodegenerative disorders Alzheimer, Parkinson and Huntington diseases7, but also depression, anxiety8, schizophrenia, autism9, stroke10 etc. characterized by transcriptional and/or post-translational modification of NMDAR10,11. NMDAR has also been found outside the CNS: it is expressed by osteoclasts, osteoblasts, pancreatic beta cells, testis, keratinocytes, renal podocytes, immune cells, skeletal muscle, cardiomyocyte, etc. and also cerebral and aortic endothelial and smooth muscle cells12-25. On an other hand, vesicular glutamate transporters (Vgluts), allowing glutamate accumulation and fast release in a calcium-dependant way, a necessary event for fast glutamatergic communication, are also identified in peripheral tissues such as bones, islets of Langerhans, testes, pineal gland, intestines and stomach26. Studies have suggested a potential role of NMDAR in the development of chronic peripheral disorders such as osteoporosis13 and type 2 diabetes mellitus15, indicating that targeting peripheral NMDAR with a specific antagonist could be beneficial in these conditions. Some cancer cells can hijack the NMDAR to proliferate in an aberrant way27. The use of NMDAR-specific antagonists in animal models of cancer has shown dramatic improvements of the animal survival breaking tumor growth28,29. Additionally it has been demonstrated that NMDAR activation of cerebral or aortic endothelial cells contributes to blood-barrier opening, monocyte infiltration, reactive oxygen species production, apoptosis or proliferation18-24. In aortic smooth muscle cells, NMDAR activation triggers proliferation and MMP-2 synthesis25.